therascreen KRAS Pyro Kit

For sequencing-based detection and quantitation of cancer-related gene mutations in the KRAS gene

Features

  • Compliance with EU IVD Directive 98/79/EC
  • Comprehensive results in real time
  • Accurate quantification of mutations in the KRAS gene
  • Easy interpretation of complex sequence information
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therascreen KRAS Pyro Kit (24)

Cat. No. / ID: 971460

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR MasterMix, CoralLoad Concentrate, and therascreen Buffers and Reagents
SEK 21,605.00
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The therascreen KRAS Pyro Kit is intended for in-vitro diagnostic use.
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Product Details

The therascreen KRAS Pyro Kit is a molecular detection kit for identification of mutations and deletions in the KRAS gene. The kit contains primers and reagents for amplification of the KRAS gene plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.

Performance

Linearity

Linearity was measured according to CLSI Guideline EP6-A “Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline”.
Plasmids carrying wild type and mutant sequences were mixed in proportions to give the following levels of mutation: 0, 12.5, 25, 37.5, and 50%. Four replicates of the mixes were placed in a random pattern on a plate and analyzed. The results for the mutation GGT->TGT in codon 12 are shown (figure " Linearity of GGT->TGT mutation").

Precision 

Determination of the linearity of the mutation GGT->TGT in codon 12 was repeated by 3 operators on 3 separate days using different combinations of PyroMark Q24 instruments and reagents.

The values for the intermediate precision (SD) were 0.6–2.0 % units in the measured range of 0–50% mutation level.

 Intermediate precision
% mutated plasmid

Run 1

(Mean, SD)  

Run 2

(Mean, SD)  

Run 3

(Mean, SD)

Summary

(Mean, SD)

0.0 0.6, 0.2 1.7, 0.7 0.7, 0.2 1.0, 0.6
12.5 13.3, 1.5 16.2, 1.9 14.6, 3.0 14.7, 1.4
25.0 23.8, 1.4 26.8, 2.4 26.9, 2.9 25.8, 1.8
37.5 42.0, 1.9 41.7, 0.5 38.5, 2.6 40.7, 2.0
50.0 49.7 2.4 50.5, 1.8 49.1, 4.8 49.8, 0.7
All values are given as % units. SD: standard deviation. % mutated plasmid based on OD260 measurement.
 

 

See figures

Principle

The therascreen KRAS Pyro Kit is used for quantitative measurements of mutations in codons 12, 13, and 61 of the human KRAS gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The KRAS gene encodes a protein that plays a critical role in the EGFR signaling cascade. Mutations in the KRAS gene can affect how the protein stimulates these downstream pathways. KRAS is mutated in approximately 30% of all cancer types. Cancers that exhibit a high frequency of KRAS mutation include colorectal cancer (35%) and lung (18%) cancer.

The following mutations are detected:

  • Codon 12/13: G12A, G12C, G12D, G12R, G12S, G12V, G13D
  • Codon 61: Q61E, Q61H, Q61L, Q61R

Procedure

The product consists of 2 assays: one for detecting mutations in codons 12 and 13 and the second for detecting mutations in codon 61 (see figures " Illustration of the KRAS assay" and " Pyrogram trace of a normal genotype in codons 12 and 13"). The two regions are amplified separately by PCR and sequenced through the defined region. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.

After PCR using primers targeting codons 12/13 and codon 61, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the corresponding sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file. The KRAS Plug-in Report should be used to analyze the run. This report ensures that the correct limit of detection (LOD) values are used and different sequences to analyze are automatically used to detect all mutations. However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System (see figure " Pyrogram trace of a GGT to GAT mutation in base 2 of codon 12"). The "Sequence to Analyze" can be then adjusted for detection of rare mutations after the run (see figures " Pyrogram trace of a GGT to TGT mutation in base 1 of codon 12" and " Pyrogram trace of the reanalysis of the sample in the previous figure").

See figures

Applications

The therascreen KRAS Pyro Kit enables quantitative detection of mutations in codons 12, 13, and 61 of the human KRAS gene. The kit is intended to be used as an aide to identify colorectal cancer patients more likely to benefit from anti-EGFR therapies.

The following mutations are detected:

  • Codon 12/13: G12A, G12C, G12D, G12R, G12S, G12V, G13D
  • Codon 61: Q61E, Q61H, Q61L, Q61R

Supporting data and figures

Resources

Analysis Software (1)
KRAS Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.6
For use with PyroMark Q24 Software version 2.0.8
Software User Guides (1)
For installation and use with PyroMark Q24 Instruments and PyroMark Q24 Software version 2.0

FAQ

Can I use more than 10ng DNA for the PCR reaction for my therascreen Pyro Kits?
It is not recommended to use more than 10ng for the PCR reaction in the therascreen BRAF, therascreen EGFR, therascreen KRAStherascreen NRAS Pyro, and therascreen UGT1A1 Kits.  Too much DNA can sometimes lead to inhibition problems in the PCR reaction.
FAQ ID -2385
Which purification kits are recommended for the therascreen Pyro kits?

 

Which purification kits are recommended for the therascreen Pyro kits?

 

FAQ ID -2386
Can PyroMark Gold reagents be vortexed?
Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.
FAQ ID -2844
What plug-ins are available for the therascreen Pyro kits?

Plug-Ins are available for:


These plug-ins are available for download on the respective catalog page under the Product Resources tab in the Analysis Software section. Note: PyroMark Q24 software version 2.0.7 is needed for the usage of the Plug-ins. 

 

FAQ ID -2387